Acid sensing by the Drosophila olfactory system

Abstract

The odour of acids has a distinct quality that is perceived as sharp, pungent and often irritating. How acidity is sensed and translated into an appropriate behavioural response is poorly understood. Here we describe a functionally segregated population of olfactory sensory neurons in the fruitfly, Drosophila melanogaster, that are highly selective for acidity. These olfactory sensory neurons express IR64a, a member of the recently identified ionotropic receptor (IR) family of putative olfactory receptors. In vivo calcium imaging showed that IR64a+ neurons projecting to the DC4 glomerulus in the antennal lobe are specifically activated by acids. Flies in which the function of IR64a+ neurons or the IR64a gene is disrupted had defects in acid-evoked physiological and behavioural responses, but their responses to non-acidic odorants remained unaffected. Furthermore, artificial stimulation of IR64a+ neurons elicited avoidance responses. Taken together, these results identify cellular and molecular substrates for acid detection in the Drosophila olfactory system and support a labelled-line mode of acidity coding at the periphery.

Figure 1. Identification of a glomerulus, DC4, activated by the CO₂ metabolite, carbonic acid

a, Behavioral testing in a T-maze at permissive (21°C) and non-permissive temperatures (29/34°C). ‘Control’ in this and all subsequent figures refer to responses of flies given a choice between two blank tubes. N=6-8. Error bars: s.e.m. ***P<0.001 ANOVA tukey-test. b, A single optical plane of the AL illustrates that the DC4- and V-glomeruli, among others, are labeled by GC16-GAL4. c, In vivo calcium imaging of the AL of a fly carrying GC16-GAL4 and UAS-GCaMP. Arrow indicates DC4. Peak ΔF responses are shown here. Scale bars: 10μm.

Figure 2. DC4 is innervated by coeloconic sensillar neurons expressing IR64a

a, Two different optical planes of the AL- DC4 (left) and DP1m (right)- of a fly bearing IR64a-GAL4; UAS-CD8GFP (a, b, c). b, IR64a+ cells in green (arrows) extend their dendrites to the 3^rd chamber of sacculus. Red autofluorescence fortuitously depicts the outline of the antenna and sacculus. I, II and III represent the 1^st, 2^nd and 3^rd chamber, respectively. c, An optical plane of the antenna across the 3^rd chamber (arrow) immunostained with α-Elav (red) and α-GFP (green). Arrowheads: dendritic terminals. Double arrowhead: axonal bundles. Scale bars: 10μm.

Figure 3. Activation of DC4 by acidity requires IR64a

a, In vivo calcium imaging of IR64a-GAL4; UAS-GCaMP flies with different genotypes. For each genotype, pre-stimulation (left), peak ΔF responses (middle) and traces for glomerular activation (right) are shown. Arrow: DC4; Arrowhead: DP1m. b, Integration of the GCaMP signals during glomerular activation. N=4-12. c, The GCaMP signal of DC4 responding to sodium acetate solution with different pH. The dotted line is a nonlinear regression fit of the data. d, α-IR64a (green) immunohistochemistry on sectioned antennae. Red auto-fluorescence outlines the antenna. Scale bars in a and d: 10μm.

Figure 4. IR64a+ neurons and IR64a are necessary and sufficient for avoidance behavior

a, Avoidance responses of flies expressing TNT or inactivated (Imp) TNT by IR64a-GAL4 in a T-maze. N=13-37. b, Calcium imaging of GR63a;IR64a^mi mutant carrying IR64a-GAL4, UAS-GR21a, UAS-GR63a and UAS-GCaMP in response to 5% CO₂. c, Avoidance of GR63a;IR64a^mi mutant expressing CO₂ receptors to 5% CO₂. d, Avoidance of flies with different genotypes blindly tested in a T-maze. N=12-27. e, α-HA immunohistochemstry on a fly harboring IR64a-HA genomic transgene. IR64a-HA protein (green) is localized in dendrites (left), but not in axons (right). Scale bars: 10μm. *** P<0.001; * P<0.05 ANOVA tukey-test.

Figure 5. Fruit flies are attracted to components other than acetic acid in vinegar

a, Calcium imaging of flies bearing IR64a-GAL4 and UAS-GCaMP. b, Integration of the GCaMP signals during odor presentation. c, Responses of starved flies to regular (pH~2.5) and neutralized (pH~7.5) vinegar in a T-maze. RWV: red wine vinegar. N=8-22. Positive avoidance index indicates an avoidance whereas negative index shows an attraction. d, Starved flies are attracted to diluted ACV (25%). N=6-11. e, Starved flies are not attracted to diluted acetic acid. The acidity of 25% ACV is same as that of 1.25% acetic acid. N=5-8.